Introduction to projects of the Laboratory The PDMI pioneered the development of a new generation of vaccines in which capsular polysaccharides (CP) are chemically bound to immunogenic carrier proteins to form conjugates. Such conjugates, which confer T-cell dependence and booster responses to polysaccharide antigens, have been successful, as exemplified by the H. influenzae type b conjugate vaccine;H. influenzae type b meningitis (the most common cause of acquired mental retardation) has been virtually eliminated wherever the vaccine has been used. PDMIs methods have now yielded conjugate vaccines against pneumococci, Salmonella typhi, nontyphoidal Salmonella, Shigella, Vibrio cholera. The prinicpes that evolved from these studies have been applied to preparing improved vaccines for anthrax and Plasmodium falciparum and vivax malaria. The Vi CP conjugate was over 90 percent protective against typhoid fever in two- to five year-olds. Based upon IgG anti-Vi levels found to be protective and of long duration in 2 to 5 year-olds, Vi-rEPA was evaluated in infants administered concurrently with DTP. No serious adverse events were observed in any of the infants, IgG anti-Vi, and levels estimated to be protective were elicited in 90% of infants when Vi-rEPA conjugate was injecred along with EPI vaccines in Vietnam. Following Phase 1 and Phase 2 studies that showed safety and immunogenicity, a doubled blinded randomized and vaccine-controlled Phase 3 evaluation of Shigella sonnei and Shigella flexneri 2a O-specific polysaccharide (O-SP)-protein conjugates, injected twice 6 weeks apart into 2,799 healthy 1 to 4 year-olds at 13 sites in Israel, were completed. The children were monitored for 48 hours after injection or for an additional 24 hours after an adverse reaction returned to normal. No serious adverse reactions related to Shigella vaccination occurred;1405 children were immunized with S. sonnei and 1343 with S. flexneri 2a conjugates. Diagnosis of shigellosis was made by demonstration of shigella in the stool of the vaccinees. Sera were taken randomly from 2 vaccinees monthly at each site and the IgG antibody measured by ELISA. Both the antibody levels and the efficacy were age related. The efficacy of the S. sonnei conjugate in the 3-4 year-olds was about 70% after 2 years of follow up (P=0.05). The incidence of S. flexneri 2a was also reduced but the numbers were too small to be significant. Accordingly, we have developed methods to increase the immunogenicity of the conjugates. This new method involves the derivatization of the KDO residue at the reducing end of the O-SP using oxime chemistry and theoretically could be applied to all lipopolysaccharides (LPS) considered for vaccines. For many reasons, Shigella and Escherichia coli are now considered as one genus. The O-specific polysaccharide (O-SP) of E. coli O157 was bound to rEPA to produce an investigational vaccine against this pathogen, which causes the often fatal hemolytic uremic syndrome, especially in small children. The investigational vaccine has proven to be safe and immunogenic in infants, and clinical efficacy trials are being planned. In an effort to produce an anthrax vaccine with fewer side effects than those associated with the licensed vaccine (AVA), a recombinant Protective Antigen (rPA), of the Bacillus anthracis toxin, was produced on an industrial scale in collaboration with researchers at the NIDDK and NIAID. It elicited in mice neutralizing antibody levels comparable to those of AVA. The rPA has been studied in adults in a Phase 1 trial. No serious adverse reactions have been noted. Post-immunization bleedings were completed in july, 2010. The data show that the rEPA elicited slightly higher antibody levels elicited by 4 injections, spaced apart as recommended by the FDA as compared to 8 injections according to the U.S. Armed Forces schedule for the licensed AVA. Because anthrax is a capsulated organism with opsonophagocytic killing, a conjugate was produced. The spore coat of B. anthracis (anthrax) contains a saccharide whose non-reducing terminus is anthrose: this saccharide is also immunodominant. A cross reactive saccharide has been detected in the CP of Shiwanella, a marine bacteria, and in the pili of Pseudomonas syringae. The Shiwanella CP has been purified and conjugated to proteins. The conjugates elicited specific antibodies that reacted with the native organism and with B. anthracis spores. Such conjugates could provide yet another antibody towards this pathogen that could add to our arsenal of immunologic defense against bioterrorism. Work is in progress to produce a conjugate vaccine against Borrelia burgdorferi, the causative agent of Lyme disease. The PDMI has coordinated long-term clinical studies in Denmark and Iceland to complement their short-term study of autoimmune diseases following systemic infection with Group B Neisseria meningitis (GBM). More recently, PDMI investigators have discovered that group C meningococcal conjugates induce group B meningococcal polysaccharide antibodies in experiment animals and human infants. Production of clinical lots of our GBM polysaccharide and the cross-reacting Escherichia coli K92 conjugates is underway. Despite the high rates of immunization with a safe acellular pertussis vaccine, pertussis continues to occur in infants too young to be fully immunized and in young adults. A Bordetella bronchiseptica strain was genetically engineered to produce the mutant pertussis toxin and culture conditions to increase its yield were devised. We proposed that such a mutant pertussis toxin should replace the licensed chemically-inactivated toxin and the other pertussis components in current vaccines. A method for synthesizing LPS-conjugates for prevention of B. pertussis and other Bordetellae has been published and is undergoing pre-clinical testing. The LOS of B. pertussis is also being explored as a protective antigen. In vitro complement-mediated reactions against this pathogen are being explored as potential vaccine. Micro-methods for testing lipid-based components of group B streptococcus are being developed in order to evaluate the effect of a high degree of asymptomatic colonization of mothers with this pathogen during delivery and of treatment with penicillin and how these relate to respiratory distress in the newborn.